CA2041304A1 - Process for the preparation of xylanase - Google Patents
Process for the preparation of xylanaseInfo
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- CA2041304A1 CA2041304A1 CA002041304A CA2041304A CA2041304A1 CA 2041304 A1 CA2041304 A1 CA 2041304A1 CA 002041304 A CA002041304 A CA 002041304A CA 2041304 A CA2041304 A CA 2041304A CA 2041304 A1 CA2041304 A1 CA 2041304A1
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- Prior art keywords
- xylanase
- fungus
- thermomyces lanuginosus
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- dsm
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01032—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/911—Microorganisms using fungi
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Abstract
Abstract Process for the preparation of xylanase by cultivation of a fungus in a nutrient medium which contains corn cobs, the use thereof, the fungus Thermomyces lanuginosus DSM 5826, an exo- and endo-cellulase-free xylanase prepare therewith, and the use thereof.
Description
3V~
Proces~ ~or the preparation of xylan~se The invent10n relates to a process for the preparation of xylanase, to the use thQreo~, to the microorg~nism T~erm~ L_~anuqinosus DS~ 5826 and to S exo- and ondocellul~e-free xylana~2 produced by the lat~er.
The breakdow~ of hemicel~ulose, w~ich is mainly . compo~ed of xylan in the ca~e of annual plan~s or deci-duous trees, is a ~tep ~hich i8 necessa~y in the produ~-tion o~ cellulo~e. Thi~ breakdo~n can be carried outeither by ch~ical means, for exam~le by hot alkaline extraction, or by enzymatic means by treatment with substrate specific enzymes, specifically with xylanase~.
Enzymatic treatment of unbleached or sem~bleached pulp wit~ xylanases results in ~reakage of hemicellulose linkage~ and breakdown of xylan. However, it is possible to employ for.this purpose only pure xylanases which contain no cellulase impurities because otherwise cellu-lose is al~o cleaved and broken down, which is extremely undesirable.
Xylanases which break down and utilize as C
source the xylan-containing raw materials present in ~he nutrien~ medium are produced by, inter alia, a number of mesophilic and thermophilic microorganisms. Howe~er, depending on the other raw materials present in the nutrient medium, predominantly cellulose, the cellulases specific for this substrate are also produced~ In order to obtain ~xo- a~d end~cellulase-free xylanase it is necessary to separate and purify the xylanase from the produced cellul ses in an elaborate process. In order to r~duce the production of cellulases during fermentation~
the microorganisms can also be cultivated on purified xylan~ The use of xylanases which have been obtained from Trichoderma harzianu~ cultivated on purified xylan for the selective breakdown of xylan is described in D. J.
Senior et al., Biotechnology Letters, Vol. 10, No. 12, p.
907-91~(1988). ~owever, cultivation on highly pure ~ylan is unsuitable ~ecau~e of the high costs of raw materials.
Proces~ ~or the preparation of xylan~se The invent10n relates to a process for the preparation of xylanase, to the use thQreo~, to the microorg~nism T~erm~ L_~anuqinosus DS~ 5826 and to S exo- and ondocellul~e-free xylana~2 produced by the lat~er.
The breakdow~ of hemicel~ulose, w~ich is mainly . compo~ed of xylan in the ca~e of annual plan~s or deci-duous trees, is a ~tep ~hich i8 necessa~y in the produ~-tion o~ cellulo~e. Thi~ breakdo~n can be carried outeither by ch~ical means, for exam~le by hot alkaline extraction, or by enzymatic means by treatment with substrate specific enzymes, specifically with xylanase~.
Enzymatic treatment of unbleached or sem~bleached pulp wit~ xylanases results in ~reakage of hemicellulose linkage~ and breakdown of xylan. However, it is possible to employ for.this purpose only pure xylanases which contain no cellulase impurities because otherwise cellu-lose is al~o cleaved and broken down, which is extremely undesirable.
Xylanases which break down and utilize as C
source the xylan-containing raw materials present in ~he nutrien~ medium are produced by, inter alia, a number of mesophilic and thermophilic microorganisms. Howe~er, depending on the other raw materials present in the nutrient medium, predominantly cellulose, the cellulases specific for this substrate are also produced~ In order to obtain ~xo- a~d end~cellulase-free xylanase it is necessary to separate and purify the xylanase from the produced cellul ses in an elaborate process. In order to r~duce the production of cellulases during fermentation~
the microorganisms can also be cultivated on purified xylan~ The use of xylanases which have been obtained from Trichoderma harzianu~ cultivated on purified xylan for the selective breakdown of xylan is described in D. J.
Senior et al., Biotechnology Letters, Vol. 10, No. 12, p.
907-91~(1988). ~owever, cultivation on highly pure ~ylan is unsuitable ~ecau~e of the high costs of raw materials.
- 2 - 2 ~
It ha~ now been found, unexpectedly, ~hat xylanase with ~u~prisingly high activity is produc~d when a fungus i~ cultivatad in a nutrient med~u~ which con-tains corn cobs, with the resulting xylanase displaying S only ~ery low or no QXO- ~nd endocellul~se act~vities.
The in~ension therefore relates to a proce~s for the pr~paration of xylanase, which i8 characterized in that a fungu~ i~ cultivated in ~ nutrient medium which co~tains corn cob~.
Xylana~e i8 produced according to the in~entiQn by culti~ation of a f~ngu~ in a nutrient medium which contain~ corn cobs. By xylanase i5 meant in thi~ context only thos~ xylanases which display only very low or no exo- or endocellulase activities. The ~ungi which can be eDployed therein are those fungi which are able to produca high xylanase activiti~e~s in the process according to the invention. One example of such fungi is Thermomvces lanuginosus. A strain of the order Mon_ iales was isolated from a pile of ~ute waste in a ~ute factory in Bangladesh in which ~ute ~ibers are treated with an oil emulsion. The temperature in the pile of ~ute waste was 65 to 70C. This strain has been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen undex DSM 5826. This strain is particularly suitahle for producing xylanase with surprisingly high activity in a nutrient medium which contains corn cobs. Therm myces lanu~L~__sus DSM 5826 is new, and the invention likewise relates to it~
The nutrient medium in which the fungus is cultivated contains corn cobs besides the nutrient5 and trace slements necessary for growth. The corn cobs can be employed as such or ground and, where appropriate, sterilized by heating to 110 to 130C or by pre~reatment with, for example, superheated steamO Particularly high xylanase activities are, surpri~ingly, achieved when the corn cobs are coarsely ground before they axe u~ed.
Although the results are still surprisingly good if the corn co~s are shredded or finely ground, the use of coarsely ground corn cobs showed surprising resnlts.
It ha~ now been found, unexpectedly, ~hat xylanase with ~u~prisingly high activity is produc~d when a fungus i~ cultivatad in a nutrient med~u~ which con-tains corn cobs, with the resulting xylanase displaying S only ~ery low or no QXO- ~nd endocellul~se act~vities.
The in~ension therefore relates to a proce~s for the pr~paration of xylanase, which i8 characterized in that a fungu~ i~ cultivated in ~ nutrient medium which co~tains corn cob~.
Xylana~e i8 produced according to the in~entiQn by culti~ation of a f~ngu~ in a nutrient medium which contain~ corn cobs. By xylanase i5 meant in thi~ context only thos~ xylanases which display only very low or no exo- or endocellulase activities. The ~ungi which can be eDployed therein are those fungi which are able to produca high xylanase activiti~e~s in the process according to the invention. One example of such fungi is Thermomvces lanuginosus. A strain of the order Mon_ iales was isolated from a pile of ~ute waste in a ~ute factory in Bangladesh in which ~ute ~ibers are treated with an oil emulsion. The temperature in the pile of ~ute waste was 65 to 70C. This strain has been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen undex DSM 5826. This strain is particularly suitahle for producing xylanase with surprisingly high activity in a nutrient medium which contains corn cobs. Therm myces lanu~L~__sus DSM 5826 is new, and the invention likewise relates to it~
The nutrient medium in which the fungus is cultivated contains corn cobs besides the nutrient5 and trace slements necessary for growth. The corn cobs can be employed as such or ground and, where appropriate, sterilized by heating to 110 to 130C or by pre~reatment with, for example, superheated steamO Particularly high xylanase activities are, surpri~ingly, achieved when the corn cobs are coarsely ground before they axe u~ed.
Although the results are still surprisingly good if the corn co~s are shredded or finely ground, the use of coarsely ground corn cobs showed surprising resnlts.
- 3 - 2~3~
Suitable nitrogen source~ are~ ~or example~ meat peptone~
fish peptone, urea, ammonium sulfate, malt extract, meat ~axtract, soya bean meal, ye~st extract and th~ like, inorganic 8alt8, for axample, pota~si~m bl~ul~ate, potassium dihy~rogen phosphate, disodium hydrogen phos-phate, iron sulfate, calcium chlcride, magnesium sulfate and the like. I~ order to increa~e the rate of release of x~lanase into ~he ~utri~n~ medium i~ may be ad~ant~geous . to add ~urface-acti~e substance~. Normally; non-ionic surface-ac~i~e substances, for example T~een 4D, Tween 60 or Tween 80, are employed in an amount of 0.05 to 0.5% by ~ight based on he total amoun~ of the medium. I~ is also po~cible, where appropriate, to add ~race elements, for example uncommon metals such as Mn~+, Zn2~, Fe2~ or vitamins, to the medium. The medium is, where appro-priate, advantageously adjus~d to a p~ of 5.0 to 8.0, preferably to 6.0 to 7.0, with ammonia or phosphoric acid.
~he fungus is culti~ated in the nutrient medium at a temperature of about 30 to 70C, preferably at 40 to 60C, particularly prefexably at 45 to 55C. It is unnecessary to keep constant the pH set at the start of the fermentation. Howaver, where appropriate, it can also be kept constant by metering in, fox example, ammonia or phosphoric acid.
After completion of the fermentation, the xylanase can be isolated from the fermentation process in a conventional manner. For ~his, for example, fungal mycelium0 spores and remaining undissolved subs~ances are 3Q removed by centrifugation or filtration. The enzyme can be further puri~ied in a conventional manner, for example by filtering out with ammonium sulfate or by solvent precipitation with acetone, alcohol or the like. The crude enzyme obtained in this way can, where appropria~e, be further purified, for example by gel filtration, ion exchange chromatography, gel electrophoresis and the like.
~7The xylanase prepared according to the invention has a high xylanase activity.It has emexged that 3~1~
surprislngly high xylanase acti~itie~ can be ~chieved on u~e of corn cobs by comparison with other untre~ted raw materlal~ such as barley 8pelt9, wheat straw, ~heat br~n, ground beech bsrk, al~alfa meal, red clover/gras3 meal, 80ya bean oil as C so~rce. The activity of the produced xylanase i5 fur~her increased by a multiple when coarsely ground corn cob3 ara employed.
The xylanase prepared according to the invention . con~ains only low or no exo- and endocellula~e acti-vi~ies~ The ~ylanase produced by Thermomyce~ lanu~inosus DSM 5~26 has proved in ~everal assays to be exo- and endocellulase-free. Such an exo- and endocellulas~-free xylanase is new, and the invention liXewise relates to it. Moreover, Thermomyces lanuqinosus DSM 5826 is able to produce this xylanase not only in a nutrient medium which contains corn cobs but also in' a nutrient ~.edium which, in place of corn cobs, contains other solid or dissolved xylan-containing carbon sources such as, for example, barley spelts, ground wheat straw, unbl ached cellulose or xylan itself. However, surprisingly high xylanase activities are obtained with corn cobs in the nutrient medium.
The exo- and endocellulose-free xylanase according to the invention had the following properties after preci-pitation ~y ethanole and lyophilization:
a) pH stability (Fig. 1) The enzyme was incubated at 20-C in buffer solu-tions at various pH values for 66 hours. The acti-vity of all trials was d~termined with 1 % hemicel~
lulose at pH 4.8 as follows: 1 ml of 1 % substrate solution in Na citrate buffer (pH 4.8), (xylan from oat spelts; Sigma X-0627) was incubated at 50~C for 2 min, and after addition of 0.5 ml of enzyme solu-tion, at 50~C fo~ a further 15 min. Subsequently 3 3s ml of dinitro salicylic acid reagent (as FPU assay-of IUPAC) and O. 5 ml of 2 . 5 N NaOH was mixed in, and the mixture was heated in a boiling waterbath fo~5 min. It was subsequently cooled quickly in a cold waterbath, and the extinction at 540 nm was measured with the blank (ci~rate buffer) as refe-rence. The extinction of the - 5 ~ 3~
enzyme 10.5 ml of enzyme -~ 1.0 ml of citrate buf~er) and of the ~ub~ra~e solution ~1 ml o~ ub~trate ~olu~ion ln c~txate bu~er) mu~t be ~ubtracted from this v~luo. Tha calibra~ion plot i~ con~tructed S using 1.0 ml o~ citra~e ~uffer and O.S ml of ~and~rd solution (containi~g O.S-1.5 mg of xylo~e~
Caiculation of ~he xylanase act~v.ity:
XU/ml = ~g of reducing sugArg (ag xyloge~est) x 0.888 97-100~ of the origlnal activ~ty were obtained in a pH range of 5 . 0-7 . O
b] Optimal p~ ~Fig. 2) The ~nz~me activity was determined by incuba~ion (50C, 15 mln) with 1~ hemicellulose suspension in lS ci~rate buffer ~pH 3.0-6.S), Tris~Cl hu~fer (pH 7.0-9.0) and phosphat~ buffer (pH 6.5-8.0).
Optimal pH range: 6.0-7.5 c) Thermal stability (Fig. 3) The enzyme 801ution was incubated in O.05 molar citrate buf~er (pH 4.8~ at temperatures of 45-60C
for 0-72 hours. The activity of the enzyme was measured with 1% hemicellulose at 50C.
Measurement a~ter 20 hours at a temperature of 45C
showed 93%, and at a temperatur~ of SO~C showed 65%, of the original activity.
d) Optimal temperature (Fig. 4) The enzyme activity was determined by incubation with a 1% hemicellulose su~pension in 0.05 molar citrate buffer at pH 4.8 for lS m.in.
Optimal temperature: 65C
e) Content of exo- and endocellulases and of B-gluco-sidase of an enzyme solution having an activity of 385 XU/ml :
i) exo- and endocellulases:
No cellulas~ activity was detected in ~he ~PU
assay of I~P~C. Furthermore, a dialycis ~lbe . (regenerated cellulose) was treated for 72 hours with the enzyme in an aqueous m~dium, and the medium .was tested or the presence of ylucose but no glucose was found~
3~
Cellul~se-containing enzymes di~sol~e the dialysis tube within a ~ew hours, by contrast~
In addition, ~he nature of the reducing sugars liberated during a lengthy incuba~ion o the e~zyme on cellulose at a pH of 6.5 was deter-min~d. The measuremen~s ware carried out in thi~ c~se ~fter 3, 7, 19 and 163 hour~. It em2rged from thi~ that in all case~ only - ~ylose, æylobioses and xylotrio~es bu~ no }O glucose had been liber~ted. A ~plica technique W~8 used to test for endocellulase activitie~
with os~ - hydrox~e~hylcellulose-~talned agar gel, no activity being found. The enzy~e was shown by all the experiment~ and assays carried out ~o be completely free of cellulase~.
The xylanase according~o the invention showed no carboxymethylcellusase activity (endo-~-1,4-cellu-lase), at a determination limit of 0.~ unit/ml, and can therefore also be employed for the production of viscose.
ii) ~-glucosidase:
O.6 ml of 0.05 molar sodium citrate buffer (pH 4.8) and 0.3 ml of enzyme solution were preincubated at 50C for 2 min. Subsequently 0.3 ml of p~nitrophenyl ~-~-glucoside (4 mg/ml of ~odium citrate buffer) was added, and the mixture was incubated at 5~C ~or 10 min. The reaction was stopped by adding 2.4 ml of 1 molar Na2C03 solution~ and tha extinction at ~05 mm was measured with the blank as reference. The activity of ~-glucosid~se was calculated as ~ollows:
IU/ml = ~ x 12 18.5 x t E ... extinction t ... reaction tLme ( minutes ) The xylanase according to the invention had ~gluco~idase activity of O.2-0.9 I~/ml.
_ 7 ~04~3 ol~
The content o~ ~-gluco~da~e, whlch cleave~
cellobio~e into glucose, ha~ ~o e~fect on ~he behavior of the xylanase according ta the inv0ntion in the produc~ion of cellulose ~ecause the absence o~ exn- and endocellulase~
mea~ that no cellulos~ bre~kdown product~
whlch o~d ~e ~ttacked by ~-glucosida9e are formed.
f) In addition, the beta-xylos~da~e, arabinosidase, acetylesterase, acetylxylan estQrase and ma~ana~e activity wa~ ~ou~d to be s 100 units/l in each case.
g) The content of soluble protein in th~ enzyme wa~
800 mg~l, the molecular weight of the main prot~in was 24 to 25 kDa and the isoelectric point of the lS main protein was 4.1.
h~ The ~u~fied ~ yne /hydroly~ed enzym~ticall~ to 11 peptide~. 4 peptides with 8, 16r 5 and 12 ~mino acids re~pectiv~ly were sequenced. It emerged from this that the investigated xylana~e was blocked at the N terminus, and thu~ it was not pos~ible to start sequenci~g from the M-terminal end, whereas xylanase isolated from Humicola _lanu~inosa ( = Thermomyces lanu~inosus) according to ~nand et al., Arch. Biochem. Biophys. 276, 546-553 (1990) contained arginine as N-terminal amino acid.
The xylanase prepared according to the invention is used for the enzymatic treatment of xylan and ligno-c~lLulose-con~aining vegetable raw materials and fi~ers composed of such raw materials and can, for example, advantageously be employed for bleaching, fo.r deinking, for refining cellulose in the product-ion of viscose or for another pretreatment such as, for ex~mple, removal of xylan before digestion.
By xylan- and lignocellulose-containing vagetable raw material~ are mean~ raw materials from decid~ous and-coniferous trees, annual plan~s, for example flax, straw, bagasse, kenaf, reeds, elephant grass etc~, but also fibers~ro~ vege~able raw materials ~uch as bleached, semibleached or unbleached pulp or wa~te paper.
- 8 - 2~)~13~
It is not necessary ~or the xylanas~ preparç~d according to th~ lnvention to be puri~ied for use, it ~uffices to remove the solid nutrient medium. After removal of the solid nutrient material, the fermentation broth can be employed as ~uch dire~tly ~or the treatment o~ fibers composed of vege~able raw materials.
When employed in the cellulose industry, it is pos~ible to treat ut~dY~2sw~l æ ~L~ ~ w~las bleached pulp with the xylanase prepared according to the invention. The treatment of the pulp result~ in hemi-OE~ nl~k~s b*ing broken. Le58 bleach i~ thus used in the subsequent bleaching pxoce~s. On treatment of semibleached pulp, the xylan remaining after the pre-lLminary bleaching is broken down, which results in cleaner and pal~r pulp5. The effect of the enzyme treat-ment is determined by means of~the kappa number (R) which indicates the content of oxidizable substances, that is to say lignin, ~or example. A higher alpha-cellulose content is reached in the bleached pulp owing to the ~ylanase treatment.
Example l:
300 ml of medium consisting of 9 g of ground and dried corn cobs, 3.3 g of meat peptone, 0.6 g ~NH4)2SO4, 0.45 g urea, 0.0~ g MgSO~.7HzO, 0.09 g CaCl2.2H2O, 4.5 g KH2PO~, 0.3 ml Tween 80, 0.3 ml S1 (1.6 g~l MnSO4.1~20; 3.45 g/l ZnSO~.7H~O; 2.0 g/l CaCl2.6H2O) and 0.3 ml S2 (5 g/l FeSO4.7H2G) were adjusted ~o a p~ of 6 and autoclaved in a shaXing flask at 128C for 60 min.
Thermomyces lanuginosus DSM 5826 preculture was inocula~ed into this medium and cultivated at 50C while shaking (140 rpm) for 4.5 days.
After ~.5 days, the mixture was filtered, and the activity of xylanase (X~/ml), ~-glu~osidase (Iufml)~
carboxymethylcellulase (CMC-ase/ml~ ~ aD glucanase (FPU/ml~ was detenmined.
;~4~3~
g XU/ml 389.7 a~ter storing 3 days at 4 C 395.0 IU/ml 0.29 FPU/ml not detectable CMC-a~e/ml not detectabl~
Ex~mple 2:
Corn cobs were a) ~hredded (size of the pi~ces of corn cob a~ove 10 mm) b~ .coarsely ground (size of the pieces o~ corn cob between 3 and 10 mm) c) finely ground (size of the pieces of corn cob below 1~ 3 mm) In each case 1000 ml of a medium consisting of 28,6g of yeast extract, 4.23 g of (NH4)2SO4, 10 g of RH2PO4, Ø3 g of FeSO4.7~2O/ 0.3 g of MgSO~.7H2O and 0.3 g of CaCl2.2H2O and 25 g of a) shredded, b) coarsely ground and c) finely ground, dried corn cobs were prepared, adjusted to a pH of 6.5 and autoclaved in a shaking flask at 121~C
for 25 minutes.
Thermomyces lanuginosus DSM 5B26 preculture was inocu-lated into these media and cultivated at 50C with shaking. After 5 days, filtration was carried out, and the xylanase activity (XU/ml) in the ~arious media was determined:
after storing 1 day at4C
Corn cobs XV/ml XU/ml Shredded 754 881 Coarsely ground 1601 15~6 Finely ground 388 423 Example 3:
The following xylanase acti~ities w~re determined in the~edi~m described in Example 2 and in the mann~r described there, ~ut using other C sources.
13~
C source Xylana~e activity (XU/ml) Corn cobs tS0% finely ground, 477 50% coarsely ground) Wheat straw 232 S Wheat bran 186 Barley 8pelt8 6 Ulva rifide (alga) 28 ~lfalfa meal 20 Red clover/gras~ meal 14 Beech bar~, ground 8 Soluble starch 4 Soya bean oil 4 Example 4:
Thermomyces lanuq~ s DSM 5826 was cultivated as in Example 1, employing 9 g~of ground wheat straw in place of ground corn cobs.
XU/ml 104.3 after storing 3 days at 4C 123.9 IU/ml 0.51 FPU/ml Not detectable CMC-ase/ml Not detectable Example 5:
The mom~ lanuginosus DSM 5826 was cultivated as in Example 1, employing barley spelts in place of 2S ground corn cobs.
XU/ml 222.9 after storing 3 days at 4~C 196.2 IU~ml 0.60 FPU/ml Not detectable CMC-ase/ml Not detectable :`
" . .
2~
Example 6:
~ h-erm~my~e~-lanuainv~u~-Ds~ $826 was ~ultiva-ted as in Example 1, employing 9 g hemicellulose (xylan) in place of ground corn robs.
XU/ml 151.55 IU/ml not detectable PPU/ml not detectable ~o CMC-ase/ml not detectable Example 7: ~r Treatment of sulfate pulp Dried sulfate pulp from ruzomberok (hardwood) was heated and shaken with water at 45C for 1.5 hours. Subsequently xylanase, prepared in analogy to ~xample 1, wa~ added and shaken at 230 rpm.
20 a) 30 g of fermentation broth b~ 90 g o~ fermen-tation broth 220 g of pulp (~=19.97) 220 g o~ pulp (K= 19.97) 3SO q of wa~er _ _ _ 290 g of water _ 100 XU enzym,e were added 300 XU enzyme were added per ~ nn of fibers per gramT of fibers Af1:er removal of the pulp by filtration, the kappa nllmber was determined.
a) b) Rappa number 17.45 13.39 .
Suitable nitrogen source~ are~ ~or example~ meat peptone~
fish peptone, urea, ammonium sulfate, malt extract, meat ~axtract, soya bean meal, ye~st extract and th~ like, inorganic 8alt8, for axample, pota~si~m bl~ul~ate, potassium dihy~rogen phosphate, disodium hydrogen phos-phate, iron sulfate, calcium chlcride, magnesium sulfate and the like. I~ order to increa~e the rate of release of x~lanase into ~he ~utri~n~ medium i~ may be ad~ant~geous . to add ~urface-acti~e substance~. Normally; non-ionic surface-ac~i~e substances, for example T~een 4D, Tween 60 or Tween 80, are employed in an amount of 0.05 to 0.5% by ~ight based on he total amoun~ of the medium. I~ is also po~cible, where appropriate, to add ~race elements, for example uncommon metals such as Mn~+, Zn2~, Fe2~ or vitamins, to the medium. The medium is, where appro-priate, advantageously adjus~d to a p~ of 5.0 to 8.0, preferably to 6.0 to 7.0, with ammonia or phosphoric acid.
~he fungus is culti~ated in the nutrient medium at a temperature of about 30 to 70C, preferably at 40 to 60C, particularly prefexably at 45 to 55C. It is unnecessary to keep constant the pH set at the start of the fermentation. Howaver, where appropriate, it can also be kept constant by metering in, fox example, ammonia or phosphoric acid.
After completion of the fermentation, the xylanase can be isolated from the fermentation process in a conventional manner. For ~his, for example, fungal mycelium0 spores and remaining undissolved subs~ances are 3Q removed by centrifugation or filtration. The enzyme can be further puri~ied in a conventional manner, for example by filtering out with ammonium sulfate or by solvent precipitation with acetone, alcohol or the like. The crude enzyme obtained in this way can, where appropria~e, be further purified, for example by gel filtration, ion exchange chromatography, gel electrophoresis and the like.
~7The xylanase prepared according to the invention has a high xylanase activity.It has emexged that 3~1~
surprislngly high xylanase acti~itie~ can be ~chieved on u~e of corn cobs by comparison with other untre~ted raw materlal~ such as barley 8pelt9, wheat straw, ~heat br~n, ground beech bsrk, al~alfa meal, red clover/gras3 meal, 80ya bean oil as C so~rce. The activity of the produced xylanase i5 fur~her increased by a multiple when coarsely ground corn cob3 ara employed.
The xylanase prepared according to the invention . con~ains only low or no exo- and endocellula~e acti-vi~ies~ The ~ylanase produced by Thermomyce~ lanu~inosus DSM 5~26 has proved in ~everal assays to be exo- and endocellulase-free. Such an exo- and endocellulas~-free xylanase is new, and the invention liXewise relates to it. Moreover, Thermomyces lanuqinosus DSM 5826 is able to produce this xylanase not only in a nutrient medium which contains corn cobs but also in' a nutrient ~.edium which, in place of corn cobs, contains other solid or dissolved xylan-containing carbon sources such as, for example, barley spelts, ground wheat straw, unbl ached cellulose or xylan itself. However, surprisingly high xylanase activities are obtained with corn cobs in the nutrient medium.
The exo- and endocellulose-free xylanase according to the invention had the following properties after preci-pitation ~y ethanole and lyophilization:
a) pH stability (Fig. 1) The enzyme was incubated at 20-C in buffer solu-tions at various pH values for 66 hours. The acti-vity of all trials was d~termined with 1 % hemicel~
lulose at pH 4.8 as follows: 1 ml of 1 % substrate solution in Na citrate buffer (pH 4.8), (xylan from oat spelts; Sigma X-0627) was incubated at 50~C for 2 min, and after addition of 0.5 ml of enzyme solu-tion, at 50~C fo~ a further 15 min. Subsequently 3 3s ml of dinitro salicylic acid reagent (as FPU assay-of IUPAC) and O. 5 ml of 2 . 5 N NaOH was mixed in, and the mixture was heated in a boiling waterbath fo~5 min. It was subsequently cooled quickly in a cold waterbath, and the extinction at 540 nm was measured with the blank (ci~rate buffer) as refe-rence. The extinction of the - 5 ~ 3~
enzyme 10.5 ml of enzyme -~ 1.0 ml of citrate buf~er) and of the ~ub~ra~e solution ~1 ml o~ ub~trate ~olu~ion ln c~txate bu~er) mu~t be ~ubtracted from this v~luo. Tha calibra~ion plot i~ con~tructed S using 1.0 ml o~ citra~e ~uffer and O.S ml of ~and~rd solution (containi~g O.S-1.5 mg of xylo~e~
Caiculation of ~he xylanase act~v.ity:
XU/ml = ~g of reducing sugArg (ag xyloge~est) x 0.888 97-100~ of the origlnal activ~ty were obtained in a pH range of 5 . 0-7 . O
b] Optimal p~ ~Fig. 2) The ~nz~me activity was determined by incuba~ion (50C, 15 mln) with 1~ hemicellulose suspension in lS ci~rate buffer ~pH 3.0-6.S), Tris~Cl hu~fer (pH 7.0-9.0) and phosphat~ buffer (pH 6.5-8.0).
Optimal pH range: 6.0-7.5 c) Thermal stability (Fig. 3) The enzyme 801ution was incubated in O.05 molar citrate buf~er (pH 4.8~ at temperatures of 45-60C
for 0-72 hours. The activity of the enzyme was measured with 1% hemicellulose at 50C.
Measurement a~ter 20 hours at a temperature of 45C
showed 93%, and at a temperatur~ of SO~C showed 65%, of the original activity.
d) Optimal temperature (Fig. 4) The enzyme activity was determined by incubation with a 1% hemicellulose su~pension in 0.05 molar citrate buffer at pH 4.8 for lS m.in.
Optimal temperature: 65C
e) Content of exo- and endocellulases and of B-gluco-sidase of an enzyme solution having an activity of 385 XU/ml :
i) exo- and endocellulases:
No cellulas~ activity was detected in ~he ~PU
assay of I~P~C. Furthermore, a dialycis ~lbe . (regenerated cellulose) was treated for 72 hours with the enzyme in an aqueous m~dium, and the medium .was tested or the presence of ylucose but no glucose was found~
3~
Cellul~se-containing enzymes di~sol~e the dialysis tube within a ~ew hours, by contrast~
In addition, ~he nature of the reducing sugars liberated during a lengthy incuba~ion o the e~zyme on cellulose at a pH of 6.5 was deter-min~d. The measuremen~s ware carried out in thi~ c~se ~fter 3, 7, 19 and 163 hour~. It em2rged from thi~ that in all case~ only - ~ylose, æylobioses and xylotrio~es bu~ no }O glucose had been liber~ted. A ~plica technique W~8 used to test for endocellulase activitie~
with os~ - hydrox~e~hylcellulose-~talned agar gel, no activity being found. The enzy~e was shown by all the experiment~ and assays carried out ~o be completely free of cellulase~.
The xylanase according~o the invention showed no carboxymethylcellusase activity (endo-~-1,4-cellu-lase), at a determination limit of 0.~ unit/ml, and can therefore also be employed for the production of viscose.
ii) ~-glucosidase:
O.6 ml of 0.05 molar sodium citrate buffer (pH 4.8) and 0.3 ml of enzyme solution were preincubated at 50C for 2 min. Subsequently 0.3 ml of p~nitrophenyl ~-~-glucoside (4 mg/ml of ~odium citrate buffer) was added, and the mixture was incubated at 5~C ~or 10 min. The reaction was stopped by adding 2.4 ml of 1 molar Na2C03 solution~ and tha extinction at ~05 mm was measured with the blank as reference. The activity of ~-glucosid~se was calculated as ~ollows:
IU/ml = ~ x 12 18.5 x t E ... extinction t ... reaction tLme ( minutes ) The xylanase according to the invention had ~gluco~idase activity of O.2-0.9 I~/ml.
_ 7 ~04~3 ol~
The content o~ ~-gluco~da~e, whlch cleave~
cellobio~e into glucose, ha~ ~o e~fect on ~he behavior of the xylanase according ta the inv0ntion in the produc~ion of cellulose ~ecause the absence o~ exn- and endocellulase~
mea~ that no cellulos~ bre~kdown product~
whlch o~d ~e ~ttacked by ~-glucosida9e are formed.
f) In addition, the beta-xylos~da~e, arabinosidase, acetylesterase, acetylxylan estQrase and ma~ana~e activity wa~ ~ou~d to be s 100 units/l in each case.
g) The content of soluble protein in th~ enzyme wa~
800 mg~l, the molecular weight of the main prot~in was 24 to 25 kDa and the isoelectric point of the lS main protein was 4.1.
h~ The ~u~fied ~ yne /hydroly~ed enzym~ticall~ to 11 peptide~. 4 peptides with 8, 16r 5 and 12 ~mino acids re~pectiv~ly were sequenced. It emerged from this that the investigated xylana~e was blocked at the N terminus, and thu~ it was not pos~ible to start sequenci~g from the M-terminal end, whereas xylanase isolated from Humicola _lanu~inosa ( = Thermomyces lanu~inosus) according to ~nand et al., Arch. Biochem. Biophys. 276, 546-553 (1990) contained arginine as N-terminal amino acid.
The xylanase prepared according to the invention is used for the enzymatic treatment of xylan and ligno-c~lLulose-con~aining vegetable raw materials and fi~ers composed of such raw materials and can, for example, advantageously be employed for bleaching, fo.r deinking, for refining cellulose in the product-ion of viscose or for another pretreatment such as, for ex~mple, removal of xylan before digestion.
By xylan- and lignocellulose-containing vagetable raw material~ are mean~ raw materials from decid~ous and-coniferous trees, annual plan~s, for example flax, straw, bagasse, kenaf, reeds, elephant grass etc~, but also fibers~ro~ vege~able raw materials ~uch as bleached, semibleached or unbleached pulp or wa~te paper.
- 8 - 2~)~13~
It is not necessary ~or the xylanas~ preparç~d according to th~ lnvention to be puri~ied for use, it ~uffices to remove the solid nutrient medium. After removal of the solid nutrient material, the fermentation broth can be employed as ~uch dire~tly ~or the treatment o~ fibers composed of vege~able raw materials.
When employed in the cellulose industry, it is pos~ible to treat ut~dY~2sw~l æ ~L~ ~ w~las bleached pulp with the xylanase prepared according to the invention. The treatment of the pulp result~ in hemi-OE~ nl~k~s b*ing broken. Le58 bleach i~ thus used in the subsequent bleaching pxoce~s. On treatment of semibleached pulp, the xylan remaining after the pre-lLminary bleaching is broken down, which results in cleaner and pal~r pulp5. The effect of the enzyme treat-ment is determined by means of~the kappa number (R) which indicates the content of oxidizable substances, that is to say lignin, ~or example. A higher alpha-cellulose content is reached in the bleached pulp owing to the ~ylanase treatment.
Example l:
300 ml of medium consisting of 9 g of ground and dried corn cobs, 3.3 g of meat peptone, 0.6 g ~NH4)2SO4, 0.45 g urea, 0.0~ g MgSO~.7HzO, 0.09 g CaCl2.2H2O, 4.5 g KH2PO~, 0.3 ml Tween 80, 0.3 ml S1 (1.6 g~l MnSO4.1~20; 3.45 g/l ZnSO~.7H~O; 2.0 g/l CaCl2.6H2O) and 0.3 ml S2 (5 g/l FeSO4.7H2G) were adjusted ~o a p~ of 6 and autoclaved in a shaXing flask at 128C for 60 min.
Thermomyces lanuginosus DSM 5826 preculture was inocula~ed into this medium and cultivated at 50C while shaking (140 rpm) for 4.5 days.
After ~.5 days, the mixture was filtered, and the activity of xylanase (X~/ml), ~-glu~osidase (Iufml)~
carboxymethylcellulase (CMC-ase/ml~ ~ aD glucanase (FPU/ml~ was detenmined.
;~4~3~
g XU/ml 389.7 a~ter storing 3 days at 4 C 395.0 IU/ml 0.29 FPU/ml not detectable CMC-a~e/ml not detectabl~
Ex~mple 2:
Corn cobs were a) ~hredded (size of the pi~ces of corn cob a~ove 10 mm) b~ .coarsely ground (size of the pieces o~ corn cob between 3 and 10 mm) c) finely ground (size of the pieces of corn cob below 1~ 3 mm) In each case 1000 ml of a medium consisting of 28,6g of yeast extract, 4.23 g of (NH4)2SO4, 10 g of RH2PO4, Ø3 g of FeSO4.7~2O/ 0.3 g of MgSO~.7H2O and 0.3 g of CaCl2.2H2O and 25 g of a) shredded, b) coarsely ground and c) finely ground, dried corn cobs were prepared, adjusted to a pH of 6.5 and autoclaved in a shaking flask at 121~C
for 25 minutes.
Thermomyces lanuginosus DSM 5B26 preculture was inocu-lated into these media and cultivated at 50C with shaking. After 5 days, filtration was carried out, and the xylanase activity (XU/ml) in the ~arious media was determined:
after storing 1 day at4C
Corn cobs XV/ml XU/ml Shredded 754 881 Coarsely ground 1601 15~6 Finely ground 388 423 Example 3:
The following xylanase acti~ities w~re determined in the~edi~m described in Example 2 and in the mann~r described there, ~ut using other C sources.
13~
C source Xylana~e activity (XU/ml) Corn cobs tS0% finely ground, 477 50% coarsely ground) Wheat straw 232 S Wheat bran 186 Barley 8pelt8 6 Ulva rifide (alga) 28 ~lfalfa meal 20 Red clover/gras~ meal 14 Beech bar~, ground 8 Soluble starch 4 Soya bean oil 4 Example 4:
Thermomyces lanuq~ s DSM 5826 was cultivated as in Example 1, employing 9 g~of ground wheat straw in place of ground corn cobs.
XU/ml 104.3 after storing 3 days at 4C 123.9 IU/ml 0.51 FPU/ml Not detectable CMC-ase/ml Not detectable Example 5:
The mom~ lanuginosus DSM 5826 was cultivated as in Example 1, employing barley spelts in place of 2S ground corn cobs.
XU/ml 222.9 after storing 3 days at 4~C 196.2 IU~ml 0.60 FPU/ml Not detectable CMC-ase/ml Not detectable :`
" . .
2~
Example 6:
~ h-erm~my~e~-lanuainv~u~-Ds~ $826 was ~ultiva-ted as in Example 1, employing 9 g hemicellulose (xylan) in place of ground corn robs.
XU/ml 151.55 IU/ml not detectable PPU/ml not detectable ~o CMC-ase/ml not detectable Example 7: ~r Treatment of sulfate pulp Dried sulfate pulp from ruzomberok (hardwood) was heated and shaken with water at 45C for 1.5 hours. Subsequently xylanase, prepared in analogy to ~xample 1, wa~ added and shaken at 230 rpm.
20 a) 30 g of fermentation broth b~ 90 g o~ fermen-tation broth 220 g of pulp (~=19.97) 220 g o~ pulp (K= 19.97) 3SO q of wa~er _ _ _ 290 g of water _ 100 XU enzym,e were added 300 XU enzyme were added per ~ nn of fibers per gramT of fibers Af1:er removal of the pulp by filtration, the kappa nllmber was determined.
a) b) Rappa number 17.45 13.39 .
Claims (10)
1. Process for the preparation of xylanase, com-prising cultivating a fungus in a nutrient medium which contains corn cobs.
2. Process according to claim 1, comprising em-ploying a Thermomyces lanuginosus as fungus.
3. Process according to claim 1, comprising em-ploying Thermomyces lanuginosus DSM 5826 as fungus.
4. Process according to claim 1, comprising cul-tivating the fungus at a temperature of 30-70°C and at a pH of 5.0 to 8Ø
5. Process according to claim 1, comprising cul-tivating the fungus at a temperature of 45 to 55°C and at a pH of 5.0 to 8Ø
6. Process according to claim 1, comprising cul-tivating Thermomyces lanuginosus DSM 5826 at a tempera-ture of 45 to 50°C and at a pH of 6.0 to 7Ø
Thermomyces lanuginosus DSM 5826.
8. Exo- and endocellulase-free xylanase obtained by cultivation of Thermomyces lanuginosus DMS 5826 in a nutrient medium.
9. Use of exo- and endocellulase-free xylanase, prepared according to claim 1, for the enzymatic treat-ment of xylan- and lignocellulose-containing vegetable raw materials and fibers composed of such raw ma-terials.
10. Use according to claim 9, comprising the exo-and endocellulase-free xylanase having been obtained by cultivation of Thermomyces lanuginosus DSM 5826.
O.Z. 942
O.Z. 942
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT0103090A AT394730B (en) | 1990-05-08 | 1990-05-08 | METHOD FOR PRODUCING EXO- AND ENDOCELLULASE-FREE XYLANASE |
| ATA1030/90 | 1990-05-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2041304A1 true CA2041304A1 (en) | 1991-11-09 |
Family
ID=3505151
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002041304A Abandoned CA2041304A1 (en) | 1990-05-08 | 1991-04-26 | Process for the preparation of xylanase |
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| Country | Link |
|---|---|
| US (1) | US5183753A (en) |
| EP (1) | EP0456033B1 (en) |
| JP (1) | JPH0787970A (en) |
| AT (2) | AT394730B (en) |
| CA (1) | CA2041304A1 (en) |
| CS (1) | CS133291A2 (en) |
| DE (1) | DE59100085D1 (en) |
| DK (1) | DK0456033T3 (en) |
| ES (1) | ES2054394T3 (en) |
| FI (1) | FI911812A (en) |
| GR (1) | GR3007689T3 (en) |
| PT (1) | PT97565B (en) |
| YU (1) | YU72891A (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5320960A (en) * | 1992-04-03 | 1994-06-14 | Genencor International, Inc. | Method of preparing solution enriched in xylanase using low molecular weight alcohol, organic salt and inorganic salt |
| US5472864A (en) * | 1984-04-19 | 1995-12-05 | Genencor International, Inc. | Method of preparing solution enriched in EG III using low molecular weight alcohol, organic salt and inorganic salt |
| EP0805856B2 (en) * | 1995-01-26 | 2009-04-01 | Novozymes A/S | Animal feed additives comprising xylanase |
| US6057438A (en) * | 1996-10-11 | 2000-05-02 | Eastman Chemical Company | Process for the co-production of dissolving-grade pulp and xylan |
| US5981233A (en) * | 1997-08-21 | 1999-11-09 | Roche Vitamins Inc. | Process for manufacturing a xylanase enzyme complex from pre-treated thin stillage of rye |
| DK2258836T3 (en) | 2004-09-10 | 2016-07-25 | Novozymes North America Inc | Methods for the prevention, elimination, reduction or destruction of biofilms |
| WO2007025328A1 (en) * | 2005-08-30 | 2007-03-08 | Protech Research Pty Ltd | EXTRACTING AND PURIFYING β 1,4-XYLANASE |
| DE102005056669A1 (en) | 2005-11-28 | 2007-05-31 | Basf Ag | Fermentative preparation of organic compounds, useful for making, e.g. amino acids or enzymes, using a starch source from which non-starch solids have not been removed, hydrolyzed and enzymatically converted to monosaccharides |
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| US4568644A (en) * | 1981-12-10 | 1986-02-04 | Massachusetts Institute Of Technology | Fermentation method producing ethanol |
| CA1272150C (en) * | 1985-12-03 | 1990-07-31 | Production of glucose syrups and purified starches from wheat and other cereal starches containing pentosans | |
| CA1280704C (en) * | 1985-12-03 | 1991-02-26 | Paul Ducroo | Production of beer |
| US4725544A (en) * | 1986-04-25 | 1988-02-16 | Tan Larry U | Method for purifying xylanase |
| WO1990001060A1 (en) * | 1988-07-27 | 1990-02-08 | Iowa State University Research Foundation, Inc. | Low molecular weight xylanase glycoprotein |
| EP1353342B1 (en) * | 2002-04-12 | 2011-08-31 | Albert Maurer | Method and device for demagnetizing objects |
-
1990
- 1990-05-08 AT AT0103090A patent/AT394730B/en not_active IP Right Cessation
-
1991
- 1991-04-15 FI FI911812A patent/FI911812A/en not_active Application Discontinuation
- 1991-04-22 YU YU72891A patent/YU72891A/en unknown
- 1991-04-23 DE DE9191106502T patent/DE59100085D1/en not_active Expired - Fee Related
- 1991-04-23 EP EP91106502A patent/EP0456033B1/en not_active Expired - Lifetime
- 1991-04-23 ES ES91106502T patent/ES2054394T3/en not_active Expired - Lifetime
- 1991-04-23 AT AT91106502T patent/ATE88497T1/en active
- 1991-04-23 DK DK91106502.7T patent/DK0456033T3/en not_active Application Discontinuation
- 1991-04-26 CA CA002041304A patent/CA2041304A1/en not_active Abandoned
- 1991-05-02 JP JP3100978A patent/JPH0787970A/en active Pending
- 1991-05-03 PT PT97565A patent/PT97565B/en not_active IP Right Cessation
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| Publication number | Publication date |
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| EP0456033A3 (en) | 1991-12-18 |
| PT97565B (en) | 1998-08-31 |
| FI911812A0 (en) | 1991-04-15 |
| GR3007689T3 (en) | 1993-08-31 |
| ATE88497T1 (en) | 1993-05-15 |
| JPH0787970A (en) | 1995-04-04 |
| EP0456033B1 (en) | 1993-04-21 |
| ATA103090A (en) | 1991-11-15 |
| FI911812A (en) | 1991-11-09 |
| US5183753A (en) | 1993-02-02 |
| DK0456033T3 (en) | 1993-05-17 |
| EP0456033A2 (en) | 1991-11-13 |
| AT394730B (en) | 1992-06-10 |
| PT97565A (en) | 1992-01-31 |
| YU72891A (en) | 1994-04-05 |
| DE59100085D1 (en) | 1993-06-03 |
| CS133291A2 (en) | 1991-12-17 |
| ES2054394T3 (en) | 1994-08-01 |
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